DE19632121C2 - Transgenic plant cells and plants with altered acetyl-CoA formation - Google Patents

Description

The present invention relates to transgenic plant cells and plants with one compared to non-transformed ones Plants altered acetyl-CoA metabolism and with a altered ability to form and recover acetyl CoA (Acetyl Coenzyme A). The change in the ability to Production and use of acetyl-CoA is achieved by the introduction and expression of a DNA sequence containing a Acetyl-CoA-hydrolase preferably a deregulated or unregulated acetyl-CoA hydrolase encoded in plant Cells. The invention also relates to the use of DNA sequences encoding an acetyl CoA hydrolase, for Increase in acetyl-CoA hydrolase activity in plant Cells, in particular for the production of transgenic Plant cells and plants, with altered ability to Formation and utilization of acetyl-CoA.

Due to the continuously increasing demand for food, which results from the constantly growing world population, one of the tasks of biotechnological research is to strive to increase the yield of crops. One way to achieve this is through the targeted genetic modification of plant metabolism. The goals are, for example, the primary processes of photosynthesis, which lead to CO ₂ fixation, the transport processes involved in the distribution of pho toassimilate within the plant, as well as metabolic pathways involved in the synthesis of storage materials, eg. As of starch, proteins, fats, oils, gums, or secondary metabolites such. As flavonoids, steroids, isoprenoids (eg., Flavorings), pigments, or polyketides (antibiotics), or of plant pathogens lead. While many applications involve the steps which either lead to the formation of photoassimilates in leaves (see also EP 0 466 995 A2 or else the formation of polymers such as starch or fructans in storage organs of transgenic plants (eg WO 94/04692 A1), there are no promising approaches describing which modifications are to be introduced in the primary metabolic pathways in order to achieve a change in the ability to form and utilize acetyl-CoA For example, utilization of acetyl-CoA is important for all those processes in the plant that require larger amounts of acetyl-CoA, such as many biochemical processes in plant cells that contain acetyl-CoA A change in the acetyl-CoA formation rate is of particular importance for the formation of starch, proteins, fats, oils, Gums, or of secondary metabolites such as flavonoids, steroids, isoprenoids (flavorings, pigments), or polyketides (antibiotics). Since plants would be useful on a large scale because of various properties for producing various of the above-mentioned substances, there is a demand for plants in which the formation and distribution of acetyl-CoA in the cells is changed so as to influence the formation of the above-described substances becomes.

Thus, the present invention is based on the object Plant cells and plants with an altered ability for the formation and utilization of acetyl-CoA as well as methods to to provide their production.

The solution of this task is by providing the In the claims designated embodiments be Semi asked.

Thus, the present invention relates to transgenic plant cells having an altered acetyl-CoA metabolism that increased cells as compared to wild-type, ie, untransformed, cells due to the expression of a foreign DNA sequence encoding a protein having acetyl-CoA hydrolase activity Acetyl-CoA hydrolase activity. The expression of such a DNA sequence leads in the transgenic plant cells to increase the intracellular acetyl-CoA-hydrolase activity. Acteyl-CoA hydrolases are enzymes that catalyze the following reaction:

Acetyl-CoA ↔ acetate + HSCoA.

It has surprisingly been found that the increase the acetyl-CoA hydrolase activity in different Compartments of plant cells is possible and thus an influence on the intracellular distribution of Metabolites is made possible. This result is so far Surprisingly, as acetyl-CoA one of the central Metabolic metabolites in plant and animal Cells whose concentration is strictly regulated (Randall and Miernyk, Methods in Plant Biochemistry Vol. 3 [ISBN 0-12-461013-7]). An intrusion into the intracellular Acetyl-CoA distribution should therefore have drastic effects to have the viability of the cells. For example there are indications that the expression of the acetyl-CoA Hydrolase from yeast in E. coli has a lethal effect. In contrast, the present invention is based on Fact that an increase in the acetyl-CoA Hydrolase activity in plant cells is indeed possible is and to beneficial properties of the plant Cells leads. For example, it has been found that the Increase in acetyl-CoA hydrolase activity in the Mitochondria in the leaves of transgenic plants to one Increasing the content of soluble sugars, such as. B. Glucose, fructose and sucrose, as well as of starch leads and for the simultaneous reduction of the content of fatty acids. That the increase in mitochondrial acetyl-CoA Hydrolase activity allows a change in the Partitioning of photoassimilates in the cells. The Acetyl-CoA-hydrolase activity in the inventive  Cells are preferably at least 50% and especially preferably by 100% compared to non-transformed Cells increased. Particularly advantageous is an increase in Enzyme activity by more than 150% compared to not transformed cells.

The increase in acetyl-CoA hydrolase activity leads to an increased concentration of acetate. In contrast to Acetyl-CoA can unregulated acetate cellular membranes penetrate. Thus it stands in other cellular Compartments in higher concentration as a substrate for the reaction catalyzed by the acetyl-CoA synthetase Acetyl-CoA synthesis available. This means that it is possible by increasing the acetyl-CoA Hydrolase activity in a compartment, intracellular Distribution of acetyl-CoA to change and thus influence on the flow of metabolites into different biosynthetic pathways to take.

In a preferred embodiment, the present invention transgenic plant cells in which the acetyl-CoA hydrolase activity in the mitochondria is increased. In plant cells, the biosynthesis of Acetyl-CoA in the mitochondria through the pyrurate Dehydrogenase multienzyme complex catalyzed reaction of Pyrurat. The increase in acetyl-CoA hydrolase activity in the mitochondria leads to an increased concentration of Acetate passing through cellular membranes into others Compartments, z. B. can diffuse into the cytosol. Here can it in turn for the synthesis of acetyl-CoA, z. B. by the Acetyl-CoA synthetase can be used.

In a further preferred embodiment, the transgenic plant cells according to the invention therefore show an increased activity of the acetyl-CoA synthetase in the cytosol. This enzyme catalyzes the following reaction

Acetate + HSCoA ↔ acetylCoA + AMP + PP _i
The increasingly formed in the cytosol acetyl-CoA can be used for example for an enhanced synthesis of isoprenoids via mevalonic acid and isopentenyl pyrophosphate (Bach, Lipids 30 (1995), 191-202).

In another preferred embodiment, the acetyl CoA hydrolase activity in the cytosol of the transgenic Plant cells increased. This will in turn be an increased Reached acetate concentration. This can for example lead to more acetate being available in the plastids and converted into acetyl-CoA in these. That would be amplifies acetyl-CoA as substrate z. B. for the Fatty acid biosynthesis or isoprenoid synthesis Available.

In a particularly preferred embodiment, the show Transgenic plant cells according to the invention with a increased acetyl-CoA hydrolase activity in the In addition, mitochondria or your cytosol increased Acetyl-CoA synthetase activity in the plastids. This can cause the transfer of acetate into the plastids and its conversion to acetyl-CoA be enhanced. This then stands, for example, to an increased extent for the Fatty acid biosynthesis available.

In principle, it is also possible to reduce the acetyl-CoA Synthetase activity in the cytosol.

In a further preferred embodiment, the plant cells described above have a reduced activity of citrate synthase in the mitochondria. This enzyme catalyzes the following reaction:

Acetyl-CoA + oxaloacetate ↔ citrate + HSCoA

By reducing the activity of this enzyme, the Acetyl-CoA used as a substrate for citrate synthesis,  more acetyl-CoA stands for by the acetyl-CoA-hydrolase catalyzed reaction available, resulting in a Increased formation of acetate leads.

Another preferred embodiment of the present invention Invention provides that in the above-described Plant cells according to the invention in the mitochondria or the activity of citrate synthase is increased in the cytosol. Such an increase in the activity of citrate synthase can lead to a change in the flow of metabolites Acetyl-CoA in a specific subcellular compartment lead, and in particular an increase of Biosynthesis of fatty acids or lipids cause.

In a further preferred embodiment of the present invention, the plant cells according to the invention described above also have a reduced activity of ATP citrate lyase in the cytosol. This enzyme catalyzes the following reaction:

Citrate + HSCoA + ATP ↔ acetyl-CoA + AMP + PP _i + oxaloacetate.

Such a reduction can lead to an increase in the causing citrate to acetyl-CoA metabolism cause a backlog of metabolites in the citrate cycle can. It would therefore be possible to increase the formation of Acetate via the endogenous acetyl-CoA hydrolase, causing the Formation of lipids and / or terpenoids can be increased.

Another embodiment of the present invention provides that the plant cells have an increased ATP Citrate lyase activity in the cytosol.

Such an increase can be increased education cytosolic acetyl-CoAs result, which for enhanced synthesis of isopentenyl pyrophosphate (IPP) and thus lead to the increased formation of terpenoids.  

The transgenic plant cells described above have due to the described altered enzyme activities a altered ability compared to wild-type cells Formation and utilization of acetyl-CoA. This can, for. B. by the determination of the changed quantities or Ratios in metabolic end products and Metabolism intermediates, as in the examples be described. In particular, you can Plant cells according to the invention are produced, which altered amounts of isoprenoids, steroids, pigments, Flavonoids, hormones, fats, oils, Proteins, gums, polyketides or substances on the involved in plant pathogen defense, or their content of soluble sugars, such as. Glucose, Fructose and sucrose, as well as being altered in strength.

Such cells can in turn advantageous starting materials for further uses. For example, these can Cells for heterologous expression of other genes with the aim the enhanced synthesis of economically relevant Serve substances. For example, DNA sequences can be introduced which are the enzymes for the synthesis of Polyhydroxyalkanoic acids (eg., PHB and PHA) encode. On this way, the increasingly formed acetyl CoA for Synthesis of such acids, which is a great economic Have meaning to be used in plants. Other Examples of economically interesting substances Polyketides, flavorings, rubber, alkaloids, isoprenoids Etc.

Of particular importance is the possibility of Expression of an acetyl-CoA-hydrolase in oil-storing Tissues of a plant, such as B. the endosperm or the Cotyledons from seeds or in other oil reservoirs Organs, the flow of the seeds or organs delivered photoassimilates in the direction of the formation of Sugars, starches, fats, pigments, isoprenoids, Polyketides, steroids, flavonoids, rubbers, fabrics, who are involved in plant pathogen defense,  Proteins, and polymers such as polyhydroxyalkanoic acids (see. z. B. Poirier et al., Bio / Technology 13 (1995), 142-150) to steer. General advantages of the cells according to the invention consist in the possibility of influence on the partitioning metabolism metabolites, in particular acetyl-CoA, on the content of metabolic end products, such. B. strength and fats, the content and composition of secondary Metabolic metabolites, the energy balance and on the Content and composition of amino acids in the cells to take.

The increase in acetyl-CoA hydrolase activity in the cells of the invention is preferably carried out by the Introduction and expression of DNA sequences containing a Encode acetyl CoA hydrolase. These DNA sequences, the one Protein with the enzymatic activity of an acetyl-CoA Encode hydrolase, both prokaryotic, especially bacterial, as well as eukaryotic DNA Be sequences, d. H. DNA sequences from plants, algae, Fungi or animal organisms or sequences that Encoding acetyl-CoA hydrolases from such organisms.

In a preferred embodiment of the invention acts it is the DNA sequences that produce an acetyl-CoA hydrolase encode sequences encoding enzymes that are present in the Comparison to normally in plant cells occurring deregulated acetyl-CoA hydrolases or are unregulated. Deregulated means that this Enzymes are not regulated in the same way as the in unmodified plant cells normally formed acetyl-CoA-hydrolase enzymes. In particular these enzymes are subject to other regulatory mechanisms, d. H. they are not going to the same extent by those in the Inhibits plant cells present inhibitors or by Metabolites allosterically regulated. Deregulated means preferably in that the enzymes have a higher activity than endogenously expressed in plant cells acetyl-CoA-hydrolases  respectively. Unregulated means in the context of this invention, that the enzymes in plant cells have no regulation subject.

These enzymes encoded by the sequences may be are both known naturally occurring enzymes, a different regulation by different Have substances as well as enzymes by Mutagenesis of DNA sequences that make known enzymes Encode bacteria, algae, fungi, animals or plants, were manufactured.

In a particularly preferred embodiment of the present invention encode the DNA Sequences proteins with the enzymatic activity of a Acetyl-CoA-hydrolase from fungi, in particular from fungi of the Genus Saccharomyces. Preference is given to DNA sequences used an acetyl-CoA-hydrolase from Saccharomyces encode cerevisiae. Such sequences are known and (See Lee et al., Journal of Biological Chemistry 265 (1990), 7413-7418 (accession number M31036)). Around the localization of acetyl-CoA-hydrolase in mitochondria ensure the plant cells, have DNA Sequences encoding mitochondrial targeting sequences, with the coding region of acetyl CoA hydrolase be merged. Such sequences are known for example, Braun et al. (EMBO J. 11 (1992), 3219-3227).

Known are next to the mentioned DNA sequence Saccharomyces cerevisiae also contains other DNA sequences Proteins with the enzymatic activity of an acetyl-CoA Hydrolase encode, for example from Neurospora crassa (See EMBL accession number M31521; Marathe et al., Molecular and Cellular Biology 10 (1990), 2638-2644) and those due to their properties also for the production of Plant cells according to the invention can be used. It must be ensured that the protein in Mitochondria or in the cytosol of the plant cell  is formed. Techniques for modifying such DNA Sequences to localize the synthesized enzymes in mitochondria and in the cytosol of plant cells to ensure are known in the art. In the case, that the acetyl-CoA-hydrolases contain sequences that contribute to Secretion or for a particular subcellular localization necessary, z. B. for localization in the extracellular Space or the vacuole, the corresponding DNA Deleted sequences. Furthermore, DNA sequences, which encode an acetyl-CoA-hydrolase, with the aid of the already known above-mentioned DNA sequences isolated from any organisms. Methods for the Isolation and identification of such DNA sequences are the person skilled in the art, for example, the hybridization with known sequences or by polymerase chain reaction using primers derived from known sequences are derived.

The enzymes encoded by the identified DNA sequences are subsequently analyzed for their enzyme activity and Regulation examined.

By introducing mutations and modifications after the Technicians know well-known techniques that can be used by the DNA Se enzymes encoded in their regulatory genes Properties are changed compared to naturally occurring in plants acetyl-CoA To obtain hydrolases den- or unregulated enzymes.

The increase in acetyl-CoA synthase, citrate synthase, or ATP citrate lyase activity in the inventive Plant cells is preferably through the introduction and Expression of DNA sequences reaches such enzymes encode. These sequences may be sequences act such enzymes from prokaryotic, especially bacterial, or eukaryotic Organisms, e.g. As plants, algae, fungi or animals, encode.  

In a preferred embodiment, such enzymes are deregulated or unregulated enzymes, as described above Related to the acetyl-CoA hydrolase explained.

These enzymes encoded by the sequences may be are both known naturally occurring enzymes, a different regulation by different Have substances as well as enzymes by Mutagenesis of DNA sequences that make known enzymes Encode bacteria, algae, fungi, animals or plants, were manufactured.

DNA sequences containing acetyl-CoA synthases from various Encoding organisms are described. In animal Organisms are z. For example, those from Macropus engenii, human, Caenorhabditis elegans and Drosophila melanogaster known (see eg GenEMBL database accession numbers L15560, D16350, Z66495 and Z46786).

In a particularly preferred embodiment of the present invention, the DNA sequences encode a Acetyl-CoA synthetase with biological properties an acetyl-CoA synthase from fungi, in particular from those of the genus Saccharomyces, and particularly preferred from Saccharomyces cerevisiae. Such sequences are for example, accessible under the GenEMBL database Access numbers Z47725, M94729, L09598, X56211 for Saccharomyces cerivisiae in particular under X76891. Possible is also the use of DNA sequences that are bacterial Encoding acetyl-CoA synthetases. Such are z. B. accessible under the accession numbers M97217, M87509 or M63968.

DNA sequences encoding a citrate synthase are known from various organisms. Sequences encoding plant citrate synthases are e.g. B. known for Arabidopsis thaliana (GenEMBL database accession number X17528; Unger et al., Plant Mol. Biol. 13 (1989), 411-418), as well as for tobacco, potato and sugar beet (see WO 95/24487 A1). Furthermore, sequences encoding animal citrate synthases are known, e.g. Porcine (accession number M21197, Evans et al., Biochemistry 27 (1988), 4680-4686). Preferably, sequences are used which encode a citrate synthase with the biological properties of a citrate synthase from bacteria, in particular E. coli, or fungi, in particular Saccharomyces cerevisiae. Various sequences encoding citrate synthases from bacteria are known e.g. Available under GenEMBL database access numbers:
M33037, Z70021, M74818, Z70017, Z70009, Z70016, L38987, Z70014, Z70022, Z70019, Z70018, Z70020, Z70012, Z70010, Z70011, Z70013, Z70015, M36338, L33409, X66112, X60513, Z73101, M29728, M17149, L41815, Z34516, M73535, L14780, X55282, L75931 and D90117. A preferred sequence is as described in Ner et al. (Biochemistry 22 (1983), 5243-5249), which encodes a citrate synthase from E. coli. Sequences encoding citrate synthases from E. coli are available, for example, under the accession numbers M28987 and M28988 (see also Wilde et al., J. Gen Microbiol 132 (1986), 3239-3251). Sequences encoding citrate synthases from fungi are available under accession numbers D63376 and D69731, those from S. cerevisiae in particular under accession numbers Z11113, Z48951, Z71255, M54982, X88846 and X00782. The latter is preferably used.

DNA sequences encoding an ATP citrate lyase are for example, known from rats (Elshourbagy et al., J. Biol. Chem. 265 (1990), 1430-1435), human (Elshourbagy et al., J. Biol. Chem. 204 (1992), 491-499), C. elegans (Wilson et al., Nature 368 (1994), 32-38) and Arabidopsis thaliana (EMBL accession numbers T13771, Z18045, Z25661 and Z26232).

For the localization of the respective enzyme in the desired compartment of the plant cell applies again, what was already mentioned above in connection with the Acetyl CoA hydrolase has been said.  

The reduction of the activity of citrate synthase or the ATP citrate lyase in the cells of the invention can by means of the methods known in the art, z. B. by Expression of an antisense RNA, a specific ribozyme or by means of a cosuppressive effect.

To the expression of the DNA sequences, the above encoded enzymes in plant cells In principle, these can in principle be under the control of any functional in plant cells Promotors are provided. The expression of said DNA In general, sequences in any tissue can be one of a kind transformed plant cell according to the invention regenerated plant and take place at any time, however, it preferably takes place in such tissues, in an altered ability to educate and exploit Of acetyl-CoA beneficial for either the growth of Plant or for the formation of ingredients within the plant is. Suitable therefore appear above all Promoters that have specific expression in one certain tissues, at a particular stage of development the plant or in a specific organ of the plant to ensure. Preferably, the DNA sequences are lost the control of promoters that are seed-specific Ensure expression. In the case of starch-storing Plants, such. As of corn, wheat, barley or other Cereals will thereby have the ability to Altered formation and utilization of acetyl-CoA, and it finds an altered synthesis of seed ingredients instead of. Especially suitable for increasing the Fatty acid biosynthesis due to increased acetyl-CoA Content in seeds of oil-forming plants such as oilseed rape, Soybean, sunflower and oil palms appear promoters, specific in the endosperm or in the cotyledons of forming seeds are active. Such promoters are z. B.  the phaseolin promoter from Phaseolus vulgaris, the USP Promoter from Vicia faba or the HMG promoter from wheat.

According to the invention, it is also advantageous for expression the DNA sequences to use promoters in Storage organs such as tubers or roots are active, for. In the storage root of the sugar beet or in the tuber the potato. In this case it comes for example expression of DNA sequences containing an acetyl-CoA Hydrolase encode a reversal of biosynthetic pathways in terms of forming more sugar or starch and one altered formation and utilization of acetyl-CoA in Direction of fatty acid biosynthesis.

Furthermore, the expression of the DNA sequences under the Kon trolls are made by promoters specific to the time the bloom induction, or activated during flowering be, or who are active in tissues, for the Flower induction are necessary. Likewise, promoters to be used only by external influences be activated controlled time, for. By light, Temperature, chemical substances (see for example WO 93/07279). For increasing the export rate of Photoassimilates from the sheet are z. B. promoters of Interest, which is a companion cell-specific expression respectively. Such promoters are known (eg the promoter the rolC gene from Agrobacterium rhizogenes).

The DNA sequences containing the enzymes described above are preferred except with a promoter with DNA sequences linked to a further increase in the Ensure transcription, for example so-called Enhancer elements, or with DNA sequences in the transcribed area and the more efficient Translation of the synthesized RNA into the corresponding Ensure protein. Such regions can be of viral Genes or suitable plant genes obtained or be prepared synthetically. You can be homologous or heterologous to the promoter used. advantageously, Furthermore, the coding DNA sequences with 3'-not  translated DNA sequences linked to the termination transcription and polyadenylation of the transcript guarantee. Such sequences are known and described, for example, the Octopinsynthasegens Agrobacterium tumefaciens. These sequences are arbitrary interchangeable.

The DNA sequences according to the invention in plant cells are introduced and expressed in the Plant cells according to the invention preferably stable in Genome integrated before. In addition to the ones described above modified enzyme activities, the inventive transgenic plant cells of non-transformed Plant cells are further distinguished by the fact that they stably integrated into the genome to have a foreign DNA whose Expression the change in acetyl-CoA hydrolase activity and optionally another of those described above Enymaktivität effected. Foreign DNA means in this case Context that the DNA either with respect to the transformed plant species is heterologous, or the DNA, if it is homologous to this, in a place in the genome is localized, in which they are in non-transformed cells does not occur. This means that the DNA in one genomic environment in which it naturally resides does not occur. Furthermore, the foreign DNA usually has the Characterized that it is recombinant, d. H. out consists of several components that in this combination in do not occur to nature.

It may be in the transgenic plant cells according to the invention basically any cells Act plant species. Of interest are both cells monocotyler as well as dicotylerous plant species, in particular Cells starch-storing, oil-storing or land economic crops such. Rye, oats, ger ste, wheat, potato, corn, rice, oilseed rape, pea, sugar beet,  Soybean, Tobacco, Cotton, Sunflower, Oil Palm, Wine, To mate, etc. or cells of ornamental plants.

The subject matter of the present invention is furthermore transgenic Plants, the transgenic plant cells according to the invention contain. Such plants can, for example be prepared by regeneration of inventive Plant cells by methods known in the art.

Plants containing cells according to the invention preferably have at least one of the following characteristics:

• (a) a reduced or increased content Fatty acids in the leaf tissue or seed tissue in the Comparison to wild-type plants;
• (b) an increased content of soluble sugars in the Leaf tissue compared to wild-type plants;
• (c) an increased content of starch in leaf tissue in Comparison to wild-type plants;
• (d) reduced growth;
• (e) forming two or more sprouts;
• (f) change in leaf color.

The invention also relates to propagation material plants according to the invention, the cells according to the invention contains. These include, for example, cuttings, seeds Fruits, rhizomes, tubers, seedlings etc.

The present invention also relates to recombinant DNA molecules which contain the following elements:

• (a) a promoter functional in plant cells, and
• (b) a DNA sequence encoding a protein with the enzymatic Encoded activity of an acetyl-CoA-hydrolase, and the so linked to the promoter that they are in plant cells into a translatable RNA can be transcribed.

The transfer of DNA molecules containing DNA sequences which code for one of the enzymes described above takes place according to the skilled person known methods, preferably under Use of plasmids, in particular such plasmids, the stable integration of the DNA molecule into the genome ensure transformed plant cells, for example binary plasmids or Ti plasmids of Agrobacterium tumefaciens system. In addition to the Aqrobacterium system come other systems for introducing DNA molecules into plant cells in question, such as. B. the so-called biolistic methods or the transformation of Pro toplasten (see Willmitzer L. (1993), Transgenic Plants, Biotechnology 2; 627-659 for an overview). Procedure for Transformation of monocotyler and dicotyler plants are in of the literature and are known in the art.

Finally, the present invention relates to the use of tion of DNA sequences containing a protein with the encode enzymatic activity of an acetyl CoA hydrolase, for expression in plant cells to produce the acetyl-CoA Increase hydrolase activity in plant cells. In particular, the invention relates to the use such DNA sequences for the production of transgene Plant cells compared to non-transformed Plant cells have an altered ability to form Sugars, starches, fats, pigments, isoprenoids, Polyketides, steroids, flavonoids, rubbers, fabrics, who are involved in plant pathogen defense, Proteins, and / or polymers such as polyhydroxyalkanoic acids respectively. The increase in acetyl-CoA hydrolase activity takes place preferably in the mitochondria or in the Cytosol of plant cells.

FIG. 1 shows a schematic illustration of the 14.39 kb plasmid Bin-mHy-Int. The plasmid contains the following fragments:
A = fragment A (528 bp) contains the EcoRI-Asp718 fragment of the promoter region of the 35S promoter of the "cauliflower mosaic virus" (nucleotides 6909 to 7437) (Frank et al., Cell 21 (1980), 285-294).
B = fragment b (109 bp) comprises a DNA fragment having the coding region of the mitochondrial target sequence of the protein of the matrix processing peptidase (MPP) from potato (Braun et al., EMBO J. 11 (1992), 3219-3227 (accession number X66284 )).
C = fragment C (189 bp) comprises a DNA fragment of the intron PIV2 from the plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet. 220 (1990), 245-250).
D '= fragment D' (170 bp) comprises a DNA fragment with the 5 'region of the coding region of the acetyl-CoA-hydrolase gene (Lee et al., Journal of Biological Chemistry 265 (1990), 7413-7418) , Nucleotides 614-784 (accession number M31036).
D "= fragment D" (1420 bp) comprises a DNA fragment having the coding region of the acetyl-CoA-hydrolase gene (Lee et al., Journal of Biological Chemistry 265 (1990), 7413-7418), nucleotides 785 until 2194 (accession number M31036).
E = fragment E (192 bp) comprises the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTi-ACH5, nucleotides 11749-11939 (Gielen et al., EMBO J. 11 (1984), 3219-3227).

Fig. 2 shows a schematic illustration of the 14.28 kb plasmid Bin-Hy-Int. The plasmid contains the following fragments:
A = fragment A (528 bp) contains the EcoRI-Asp718 fragment of the promoter region of the 355 promoter of the "cauliflower mosaic virus" (nucleotides 6909 to 7437) (Frank et al., Cell 21 (1980), 285-294).
C = fragment C (189 bp) comprises a DNA fragment of the intron PIV2 from the plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet. 220 (1990), 245-250).
D '= fragment D' (170 bp) comprises a DNA fragment having the 5 'region of the coding region of the acetyl-CoA-hydrolase gene (Lee et al., Journal of Biological Chemistry (1990) 265, 7413-7418). , Nucleotides 614-784 (accession number M31036).
D "= fragment D" (1420 bp) comprises a DNA fragment having the coding region of the acetyl-CoA-hydrolase gene (Lee et al., Journal of Biological Chemistry (1990) 265, 7413-7418), nucleotides 785 until 2194 (accession number M31036).
E = fragment E (192 bp) comprises the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTi-ACH5, nucleotides 11749-11939 (Gielen et al., EMBO J. 11 (1984), 3219-3227).

Fig. 3 shows a Western blot to detect the expression of the acetyl-CoA hydrolase from Saccharomyces cerevisiae in transgenic tobacco leaves,

FIG. 4 shows three transgenic MB-Hy1 lines in comparison to a control plant (left), FIG.

FIG. 5 shows leaves of the transgenic MB-Hy1 lines 39, FIG.

Fig. 6 shows leaves of a control plant,

Fig. 7 is a plant a transgenic MB-Hy1 shows line with flowers,

Fig. 8 shows a control plant with flowers,

Fig. 9 shows a schematic illustration of the 14, 25 kb plasmid pTCSAS
A = fragment A (528 bp) contains the EcoRI-Asp718 fragment of the promoter region of the 35S promoter of the "cauliflower mosaic virus" (nucleotides 6909 to 7437) (Frank et al., Cell 21 (1980), 285-294).
B = fragment B (1747 bp) comprises a DNA fragment having the coding region of the citrate synthase gene from tobacco in reverse orientation (nucleotides 1 to 1747) (accession number X84226).
C = fragment C (192 bp) comprises the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTi-ACH5, nucleotides 11749-11939 (Gielen et al., EMBO J. 11 (1984), 3219-3227).

methods 1. Cloning procedure

For cloning into E. coli the vectors pUC9-2, pA7 (by Schaewen, A. (1989) Dissertation, Free University of Berlin) and pAM (see Example 1) det. For the plant transformation, the gene con in the binary vector pBinAR-Hyg (Accession Number: DSM 9505; 20.10.1994) cloned.

2. Bacterial strains

For the pUC vectors and for the pBinARHyg constructs E. coli strain DH5α (Bethesda Research Laboratories, Gaithersburg, USA).

Transformation of plasmids into potato plants was using the Agrobacterium tumefaciens strain C58C1 pGV2260 (Deblaere et al., Nucl. Acids Res. 13 (1985), 4777-4788).  

3. Transformation of Agrobacterium tumefaciens

The transfer of the DNA was carried out by direct transforma tion according to the method of Höfgen and Willmitzer (Nucleic Acids Res. 16 (1988), 9877). The plasmid DNA transfor mated agrobacteria was determined by the method of Birnboim and Doly (Nucleic Acids Res. 7 (1979), 1513-1523) isolated and after appropriate restriction cleavage analyzed by gel electrophoresis.

4. Transformation of tobacco

The transformation of tobacco took place after that in Rosahl et al. (EMBO J. 6 (1987), 1155-1159) Method.

5. Transformation of potatoes

Ten small leaves wounded with a scalpel Potato sterile culture (Solanum tuberosum L. cv. Désirée) were dissolved in 10 ml of MS medium (Murashige and Skoog, Physiol. Plant 15 (1962), 473) with 2% sucrose ge which gives 50 μl of a selection grown Agrobacterium tumefaciens overnight culture. To 3-5 minutes of gentle shaking was followed by another Incubation for 2 days in the dark. Then the Leaves for callus induction on MS medium with 1.6% Glu cose, 5 mg / l naphthylacetic acid, 0.2 mg / l benzylaminopu rin, 250 mg / l claforan, 3 mg / l hygromycin and 0.80% Bacto Agar laid. After one week incubation at 25 ° C and 3000 lux became the leaves for shoot induction MS medium with 1.6% glucose, 1.4 mg / l zeatinribose, 20 mg / l naphthylacetic acid, 20 mg / l gibberellic acid, 250 mg / L claforan, 3 mg / L hygromycin and 0.80% Bacto agar placed.  

6. Planting

Potato plants were kept in the greenhouse under the following conditions:

• - light period: 16 h at 25,000 lux and 22 ° C,
• - dark period: 8 h at 15 ° C
• - Humidity: 60%.

Tobacco plants were kept in the greenhouse under the following conditions:

• Light period 14 h at 10000 lux and 25 ° C,
• - dark period. 10 hours at 20 ° C,
• - Humidity: 65%.

7. Determination of acetyl-CoA hydrolase activity in Leaves of tobacco and potato plants

To determine the acetyl CoA hydrolase activity in tobacco and potato leaves, leaf samples were incubated in extraction buffer (50 mM Hepes-KOH pH 7.5; 5 mM MgCl ₂ ; 1 mM EDTA; 1 mM EGTA; 10 mM DTT; Vol./vol.) Glycerol; 0.1% (v / v) Triton X-100). After centrifugation, the cell-free extracts were used for enzyme activity measurement.
Reaction buffer: 100 mM Na-phosphate, pH 7.2 [1- ¹⁴ C] acetyl-CoA 0.5 uCi / 100 ul,
Reaction volume: 105 μl,
Reaction temperature: 30 ° C,
Measuring times: 2, 4, 6, 10 min.

principle

Are used DE81 filters (ion exchanger) wherein HSCoA and [1- ¹⁴ C bond) acetyl-CoA, however, [1- ¹⁴ C) acetate (when washing is eluted 2 times 5 min), the filter with 2% acetic acid. To determine the acetyl-CoA hydrolase activity, the radioactivity remaining on the filters was determined by scintillation measurement and comparison with calibration curves. (Roughan, PG et al., Analytical Biochemistry 216 (1994), 77-82).

8. Western blot analysis to detect the expression of Acetyl-CoA-hydrolase in leaves

To detect the acetyl CoA hydrolase in tobacco and potato leaves, leaf samples were extracted into extraction buffer (50 mM Hepes-KOH pH 7.5, 5 mM MgCl ₂ , 1 mM EDTA, 1 mM EGTA, 10 mM DTT, 10% (vol ./Vol.) Glycerol; 0.1% (v / v) Triton X-100). After centrifugation, aliquots (10 μg protein) of the cell-free extracts were used for a Western blot. Western blot analyzes were carried out using a polyclonal antibody directed against the acetyl-CoA-hydrolase from Saccharomyces cerevisiae as described in Landschütze et al. (EMBO J. 14 (1995), 660-666).

9. Determination of metabolic intermediates in tobacco leaves

The determination of sucrose, glucose, fructose and Starch was spectrophotometrically coupled enzymatic reactions according to Stitt et al. (Methods in Enzymology, 174, 518-552).

a) Determination of sucrose glucose and fructose

In each case three leaf discs with a diameter of 1.1 cm each with 500 .mu.l of 80% ethanol in water extracted at 70 ° C for 90 min. After separation of the solid from the liquid phase by centrifugation,  the liquid phase was taken off and taken for measurement the soluble sugar used.

The reaction buffer contained:
100 mM imidazole pH 6.9;
5 mM MgCl ₂ ;
2mM NADP;
1mM ATP;
2 U / ml glucose-6-phosphate dehydrogenase
G6P determination: + 1.4 U / ml hexokinase
F1P determination: + 1.4 U / ml phosphoglucoisomerase
G1P determination: + 2.0 U / ml phosphoglucomutase.

The measurement is carried out at 30 ° C with 50 ul extract.
(G6P = glucose-6-phosphate; F1P = fructose-1-phosphate;
G1P = glucose-1-phosphate)

b) Determination of starch

In each case three leaf discs with a diameter of 1.1 cm each with 500 .mu.l of 80% ethanol in water extracted at 70 ° C for 90 min. After separation of the solid from the liquid phase by centrifugation, the liquid phase was removed, the solid The residue was washed twice with 80% ethanol in water washed.

The pellet was charged with 400 μl of 0.2 N NaOH at 95 ° C an hour unlocked. It was washed with 70 μl of 1 N Neutralized acetic acid at room temperature and the solid from the liquid phase by centrifugation separated.

The starch was made using a range for the determination of starch (Boehringer, Mannheim) Manufacturer information using amyloglucosidase hydrolyzes and the liberated glucose enzymatically certainly.

c) Determination of fatty acids in leaves and seeds

Leaf discs (each 1.1 cm in diameter) or one tobacco seed each was added to 1 ml of 1N HCl in Methanol at 80 ° C under a nitrogen atmosphere after Add 5 μg of meristylic acid as an internal standard for 15 minutes in a sealed glass jar heated. After cooling to room temperature, 1 ml of 0.9% aqueous NaCl solution and 1 ml of n-hexane (p.A.) added. After extraction of the aqueous phase was the organic phase removed and gaseous Concentrated nitrogen.

The separation and quantification of Fatty acid methyl ester was carried out by Gas chromatography according to Browse et al. (Analytical Biochemistry 152 (1986), 141-145).

d) determination of chlorophyll a and b, antheraxanthin, Zeaxanthin and Violaxanthin

Leaf discs (each 1.1 cm in diameter) were directly after sampling in liquid nitrogen frozen. The following steps were included darkened room light performed.

The samples were incubated with 250 μl of ice cold 85% acetone in Homogenized water, the suspension was then Flushed with nitrogen gas for 30 seconds and then stored for 15 minutes on ice. To Centrifugation for 15 minutes at 4 ° C and 10,000 g the supernatant was passed through a Millipore Millex GV4 Sterile filter attachment filtered and then 30 Purged with nitrogen gas at 4 ° C for 2 seconds. The Samples were then measured at -70 ° C until measurement stored. The separation and quantification of Pigments were made by high pressure liquid Chromatography (HPLC) as by Zhayer and Björkman (J. Chromatogr. 543 (1990), 137-145).

The separation column was a ZORBAX ODS 5 μm non endcapped 250.4.5 mm reversed-phase column used. The detection of the pigments was carried out by  Measurement of absorbance at 450 nm in one area of 0.04 absorbance units (AUFS).

10. Determination of citrate synthase activity in leaves of tobacco plants

To determine citrate synthase activity in tobacco leaves, leaf samples were incubated in extraction buffer (50 mM Hepes-KOH pH 7.5, 5 mM MgCl ₂ , 1 mM EDTA, 1 mM EGTA, 10% (v / v) glycerol, 0 , 1% (v / v) Triton X-100; 4 mU / ml α ₂ -macroglobulin). After centrifugation, the cell-free extracts were used for enzyme activity measurement.

The activity of citrate synthase was determined photometrically:
Reaction buffer:
0.1 M Tris-Cl, pH 8.0,
0.1 mM 5,5'-dithio-bis (2-nitrobenzoic acid),
0.3mM acetyl-CoA;
Reaction volume: 700 μl,
Reaction temperature: 30 ° C,
protein used: ~60 μg,
Start: 7 μl of 50 mM oxaloacetate.

The measurement of the absorption took place at 412 nm.

The examples illustrate the invention.

example 1 Construction of the binary plasmid Bin-mHy-Int

For plant transformation, the coding region of the acetyl-CoA-hydrolase gene from Saccharomyces cerevisiae was determined by means of the polymerase chain reaction (PCR) starting from genomic Saccharomyces cerevisiae DNA by means of the primers
AcCoHyl (5'-GTCAGGATCCATGACAATTTCTAATTTGTTAAAGCAGAGA-3 ') (Seq ID No. 1) and
AcCoHy2 (5'-GTCAGGATCCCTAGTCAACTGGTTCCCAGCTGTCGACCTT-3 ') (Seq ID No. 2)
amplified. The sequence of the acetyl-CoA-hydrolase from Saccharomyces cerevisiae is registered in the GenEmbl database with the accession number M31036. The cloning of the acetyl-CoA-hydrolase gene is described in Lee et al. (Journal of Biological Chemistry 265 (1990), 7413-7418). The amplified fragment corresponds to the region from nucleotides 614 to 2194 of this sequence (accession number M31036). In each case a BamHI site was inserted at the 5'-end and at the 3'-end. The 1590 bp BamHI cut PCR fragment was cloned via the additional interfaces into the BamHI site of the vector pUC9-2. The intron PIV2 (189 bp) from the plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet. 220 (1990), 245-250) via PCR using the primer GUS-1 (5'-gtatacgtaagtttctgcttctac-3 ' (Seq ID No. 3) and GUS-2 (5'-gtacagctgcacatcaacaaattttgg-3 ') (Seq ID No. 4), rescircuited with SnaBI and PvuII, and into the singular BbrPI site of the acetyl-CoA hydrolase gene from Saccharomyces cerevisiae cloned. The correct orientation of the inserted intron (5 'end of the intron oriented towards the 3' end of the 5 'exon) was confirmed by sequence analysis. The plasmid prepared in this way was named pUC-HyInt.

The plasmid pUC-HyInt was cut with BamHI and the 1779 bp long acetyl-CoA-hydrolase fragment (with inserted intron) into the BamHI site of the plasmid pAM cloned. The plasmid thus obtained received the Designation pAM-HyInt.

The plasmid pAM was prepared as described below. The mitochondrial target sequence (111 bp) of the potato matrix processing peptidase (HPP) protein was amplified using the primers Mito-TP1 (5'-GATC GGTACC ATGTACAGATGCGCATCGTCT-3 ') (Seq ID No. 5) and Mito-TP2 (5'-GTAC GGATCC CTTGGTTGCAACAGCAGCTGA-3 ') (Seq ID No. 6) were amplified by PCR. The template used for the PCR was the plasmid pMPP (Braun et al., EMBO J. 11 (1992), 3219-3227). The amplified fragment corresponds to the region from nucleotides 299 to 397 of the MPP cDNA (Braun et al., EMBL Accession Number: X66284). An Asp718 site was inserted at the 5 'end and a BamHI site at the 3' end. The PCR fragment was cleaved with Asp718 and BamHI and the resulting 109 bp fragment subsequently cloned into the Asp718 and BamHI cleaved vector pA7 (by Schaewen, A. (1989) Dissertation, Freie Universität Berlin).

The plasmid pAM-HyInt was cut with Asp718 and XbaI and the 1887 bp fragment consisting of the coding region for the targeting peptide of the potato "matrix processing peptidase", and the coding region for the acetyl-CoA-hydrolase from Saccharomyces cerevisiae (with inserted intron) isolated and filled the 5 'overhangs of this fragment using the T4 DNA polymerase to blunt ends. The fragment thus prepared was cloned into the SmaI site of the binary plasmid pBinAR-Hyg. The coding regions of the potato matrix processing peptidase targeting peptide were oriented towards the cauliflower mosaic virus 35S RNA promoter. From this resulted the plasmid Bin-mHy-Int (see Fig. 1) which was used for the transformation of tobacco (Nicotiana tabacum SNN) and potato (Solanum tuberosum L. cv. Désirée) as described above.

Example 2 Construction of the binary plasmid Bin-Hy-Int

The BamHI fragment of the plasmid pUC-Hy-Int (see Example 1) was cloned into the BamHI site of plasmid pA7 (by Schaewen, A. (1989) Dissertation, Freie Universität Berlin). The 5 'end of the coding region of the acetyl CoA hydrolase was oriented towards the 35S RNA promoter. The plasmid thus prepared was named pA7-Hy-Int. The plasmid pA7-Hy-Int was cut with KpnI and XbaI, the 1778 bp fragment consisting of the coding region for the acetyl-CoA-hydrolase from Saccharomyces cerevisiae isolated (with inserted intron) and then into the cut with KpnI and XbaI binary plasmid pBinAR-Hyg (accession number: DSM 9505, filed: 20.10.1994). This resulted in the plasmid Bin- Hy-Int (see Fig. 2), which was used for the transformation of tobacco (Nicotiana tabacum SNN) and potato (Solanum tuberosum L. cv. Désirée).

Example 3 Analysis of transgenic tobacco plants containing an acetyl-CoA Expressing hydrolase from Saccharomyces cerevisiae

From tobacco plants transformed with the plasmid Bin-mHy-Int (see Example 1), whole tobacco plants were regenerated, transferred to soil and analyzed for the presence of Saccharomyces cerevisiae acetyl-CoA-hydrolase by Western blot analysis Browse selected. Several genotypes were identified that clearly express the acetyl-CoA-hydrolase from Saccharomyces cerevisiae (see FIG. 3). Several of the selected transgenic lines were analyzed for acetyl CoA hydrolase activity in leaves. Up to three times higher specific acetyl-CoA hydrolase activity was measured in some lines compared to the control plants (eg MB-Hy1-39, MB-Hy1-78, MB-Hy1-81, see Table I).

Table I

plant Acetyl-CoA hydrolase activity (nmol min ^-1 mg ^-1 protein control 1.49 ± 1.06 MB-Hy1-39 5.58 ± 1.59 MB-Hy1-78 2.92 ± 0.74 MB-Hy1-81 3.25 ± 0.79

The enzyme activities shown here are the mean value of at least eight measurements from at least three independent plants of said transgenic line.

The above genotypes MB-Hy1-39, MB-Hy1-78, MB-Hy1-81 were amplified, and in each case 3 plants were in one Greenhouse transferred.

Surprisingly, it was found that the leaves of Transgenic lines MB-Hy1-39, MB-Hy1-78, MB-Hy1- 81 reduced amounts of fatty acids compared to Control plants (see Table II).  

Table II

The given values represent mean values as well as the Standard deviations from 2 independent measurements represents.

Surprisingly, it was also found that the seeds the transgenic plants MB-Hy1-39, MB-Hy1-78, MB-Hy1-81 equal amounts of fatty acids compared to Control plants (see Table III).  

Table III

The given values represent mean values as well as the Standard deviations from at least 6 independent measurements represents.

Analysis of the weight of 200 seeds each showed that no significant difference between the seeds of transgenic plant MB-Hy1-39 and seeds from control plants exist (see Table IV).

Table IV

The analysis of soluble sugars such as glucose, fructose and Surprisingly, sucrose showed that the leaves of Transgenic lines MB-Hy1-39, MB-Hy1-78, MB-Hy1- 81 increased levels of sucrose, glucose and fructose  both at the end of the light phase (see Table V), and at End of the dark phase (see Table VI).

Table V

The given values represent mean values as well as the Standard deviations from 6 independent measurements represents.

Table VI

The given values represent mean values as well as the Standard deviations from in each case 3 independent measurements represents.

In the analysis of strength was continued surprisingly found that the leaves of Transgenic lines MB-Hy1-39, MB-Hy1-78, MB-Hy1- 81 increased levels of starch both at the end of the light phase, as well as at the end of the dark phase (see table VII).

Table VII

The given values represent mean values as well as the Standard deviations from 3 and 6, respectively (see Table VII) independent measurements.

When growing the transgenic plants MB-Hy1-39, MB-Hy1-78, MB-Hy1-81 in the greenhouse, it was further found that the transgenic plants had an altered phenotype compared to control plants. In particular, in the case of transgenic plants, reduced growth, the formation of multiple shoots, and a mosaic-like change in leaf coloration were noted (see FIGS. 4 and 5). For a more detailed analysis of leaf color the contents of chlorophyll a and b as well as the carotenoids zeaxanthin, antheraxanthin and violoxanthin were determined (see Tables VIII and IX).

Table VIII

The given values represent mean values as well as the Standard deviations from in each case 3 independent measurements represents.

Table IX

The given values represent mean values as well as the Standard deviations from in each case 3 independent measurements represents.  

Example 4 Construction of the binary plasmid pTCSAS

By in vivo excision from a λ ZAP II cDNA Library from Nicotiana tabacum L. obtained plasmid pTCS, contains in the EcoR I interface of pBluescript SK Vector a 1747 bp cDNA fragment with the coding region of the Citrate synthase gene from tobacco (accession number X84226).

The BamHI fragment of the plasmid pTCS was inserted into the BamHI / SalI sites of the binary plasmid BinAR cloned. The 3'-end of the coding region of the citrate Synthase oriented towards the 35S RNA promoter. That so plasmid obtained was called pTCSAS.

pTCSAS has been used for the transformation of tobacco (Nicotiana tabacum SNN).

Example 5 Analysis of transgenic tobacco plants with reduced citrate Synthase activity

Regenerated tobacco plants infected with the plasmid pTCSAS were transformed into earth and by measuring citrate synthase activity in leaves selected. Several genotypes were identified clearly a reduction in citrate synthase activity (see Table X). Several of the selected transgenic Lines were analyzed for citrate synthase activity in Scrolls analyzed. It was in some lines one order up to 6-fold decreased specific citrate synthase Activity as compared to control plants (e.g. TCSAS-14, TCSAS-17; TCSAS-26; TCSAS-43; TCSAS-48; see. Table X).  

Table X

The enzyme activities shown here are the mean value of at least 18 measurements from at least nine independent plants.

The above genotypes were TCSAS-17 and TCSAS-26 amplified, and each 6 plants were in a greenhouse house transferred.

Surprisingly, it was found that the seeds of Transgenic plants TCSAS-17 and TCSAS-26 reduced amounts contained in fatty acids compared to control plants (see Table XI).  

Table XI

The given values represent mean values as well as the Standard deviations from 10 independent measurements represents.

Analysis of the weight of 200 seeds each showed that a significant difference between the seeds of Transgenic plants TCSAS-17 and TCSAS-26 compared to the Seeds of control plants (see Table XII).

Table XII

sequence Listing

Claims (22)

1. Transgenic plant cell with an altered acetyl CoA metabolism due to the expression of a foreign DNA sequence encoding a protein with acetyl-CoA Hydrolase activity encoded, one compared to Wild-type cells enhanced acetyl-CoA hydrolase activity having. 2. Transgenic plant cell according to claim 1, wherein the Acetyl-CoA hydrolase activity in the mitochondria is increased. 3. Transgenic plant cell according to claim 2, further an increased acetyl-CoA synthetase activity in the cytosol having. 4. Transgenic plant cell according to claim 1, wherein the Acetyl-CoA hydrolase activity in the cytosol is increased. 5. Transgenic plant cell according to claim 2 or 4, which furthermore, an increased acetyl-CoA synthetase activity in the plastids. 6. Transgenic plant cell according to one of claims 1 to 5, which continues to have a reduced citrate Has synthase activity in the mitochondria. 7. Transgenic plant cell according to one of claims 1 to 5, which also has increased citrate synthase activity in the mitochondria or the cytosol. 8. Transgenic plant cell according to one of claims 1 to 7, which continues to show a decreased ATP activity. Citrate lyase in the cytosol has.   9. Transgenic plant cell according to one of claims 1 to 7, which continues to increase ATP activity. Citrate lyase in the cytosol has. 10. Transgenic plant cell according to one of claims 1 to 9, wherein the acetyl-CoA hydrolase is an unregulated or deregulated enzyme. 11. Transgenic plant cell according to claim 10, wherein the Acetyl-CoA-hydrolase from fungal cells. 12. Transgenic plant cell according to claim 11, wherein the Acetyl-CoA-hydrolase from Saccharomyces cerevisiae comes. 13. Transgenic plant cell according to claim 10, wherein the Acetyl-CoA-hydrolase from a prokaryotic Organism is derived. 14. Transgenic plant cell according to one of claims 3 or 5 to 13, wherein the acetyl-CoA synthetase a unregulated or deregulated enzyme. 15. Transgenic plant cell according to claim 14, wherein the Acetyl-CoA synthetase from a fungus, a bacterial or an animal organism. 16. Transgenic plant cell according to claim 15, wherein the Acetyl-CoA synthetase from Saccharomyces cerevisiae comes. 17. Plant containing plant cells according to one of Claims 1 to 16. 18. Plant according to claim 17, which has at least one of the following features:

• (a) a reduced or increased content of fatty acids in leaf tissue or seed tissue compared to wild-type plants;
• (b) an increased content of soluble sugars in leaf tissue compared to wild-type plants;
• (c) an increased level of starch in leaf tissue compared to wild-type plants;
• (d) reduced growth;
• (e) forming two or more sprouts;
• (f) change in leaf color.

19. Propagating material of a plant according to claim 17 or 18, the transgenic plant cells after one of the Claims 1 to 16 contains. 20. Propagating material according to claim 19, which is a fruit, a seed or a tuber is. 21. Use of DNA sequences containing a protein with the enzymatic activity of an acetyl-CoA-hydrolase encode for expression in plant cells to the Activity of acetyl-CoA-hydrolase in plant Increase cells. 22. Use according to claim 21, wherein the acetyl-CoA Hydrolase activity in the cytosol is increased.